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Arabidopsis SHORT INTEGUMENTS 2 is a mitochondrial DAR GTPase

Theresa A. Hill1, Jean Broadhvest2, Robert K. Kuzoff3, and Charles S. Gasser4

Section of Molecular and Cellular Biology, University of California, Davis, CA, 95616

1Current address: USDA ARS WRRC-GGD, 800 Buchanan St., Albany, CA 94710 2Current address: Bayer BioScience N.V., Technologiepark 38, 9052 Ghent, Belgium 3Current address: Department of Biological Sciences, University of Wisconsin, Whitewater, WI, 53190 4Corresponding author: Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Ave., Davis, CA 95616, Tel. 530-752-1013. Fax 530-752-3085 E-mail csgasser@ucdavis.edu. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AY254472.

Genetics: Published Articles Ahead of Print, published on July 18, 2006 as 10.1534/genetics.106.060657

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Running title: SHORT INTEGUMENTS2 is a mitochondrial GTPase

Key words: growth, ovule, cell division, plant development, signal transduction

Charles S. Gasser

Section of Molecular and Cellular Biology

University of California, Davis

1 Shields Ave. Davis, CA 95616

Tel. 530-752-1013

Fax 530-752-3085

e-mail : csgasser@ucdavis.edu

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ABSTRACT

The Arabidopsis short integuments 2-1 (sin2-1) mutant produces ovules with short

integuments due to early cessation of cell division in these structures. SIN2 was isolated

and encodes a putative GTPase sharing features found in the novel DAR GTPase family.

DAR proteins share a signature DAR motif and a unique arrangement of the four

conserved GTPase G motifs. We find that DAR GTPases are present in all examined

prokaryotes and eukaryotes, and that they have diversified into four paralogous lineages

in higher eukaryotes. Eukaryotic members of the SIN2 clade of DAR GTPases, have

been found to localize to mitochondria and are related to eubacterial proteins that

facilitate essential steps in biogenesis of the large ribosomal subunit. We propose a

similar role for SIN2 in mitochondria. A sin2 insertional allele has ovule effects similar

to sin2-1, but more pronounced pleiotropic effects on vegetative and floral development.

The diverse developmental effects of the mitochondrial SIN2 GTPase support a

mitochondrial role in the regulation of multiple developmental pathways.

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INTRODUCTION

Arabidopsis ovules are a useful model system for studying developmental mechanisms.

Arabidopsis ovules initiate on the inner surface of immature carpels as relatively

featureless finger-like primordia. As they elongate, three different regions become

specialized and undergo morphogenesis and cellular differentiation (ROBINSON-BEERS et

al. 1992). The distal-most region, the nucellus, is the site of meiosis and embryo sac

formation. The central chalazal region is the site of the most visible morphogenic changes

as it gives rise to two appendages, the inner and outer integuments. The basal region

elongates through division and coordinated expansion of cells forming the funiculus, a

supporting stalk. During this process, the developing ovule becomes bilaterally

symmetrical as a result of differential growth that causes the funiculus to curve toward

the base of the carpel and the outer integument to curve toward the carpel apex. At

maturity, both integuments have grown to enclose the nucellus and form a terminal

micropylar opening (Figure 1A). Mutations altering ovule morphogenesis may also

disrupt other plant developmental pathways and the relative morphological simplicity of

ovules can facilitate overall understanding of the underlying biochemical or molecular

processes.

Numerous genes affecting growth and patterning of ovules have been identified via

mutagenesis and cloning (SCHNEITZ 1999; SKINNER et al. 2004). Several of the genes

regulating Arabidopsis ovule development manifest their effects through alterations in the

pattern or progress of cell division. These genes encode proteins with a variety of

biochemical functions (SKINNER et al. 2004). For example, mutations in

AINTEGUMENTA (ANT) and INNER NO OUTER (INO), encoding AP2 and YABBY-

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domain transcription factors, respectively, result in a complete absence of both

integuments or only the outer integument (BAKER et al. 1997; ELLIOTT et al. 1996;

GAISER et al. 1995; KLUCHER et al. 1996; VILLANUEVA et al. 1999). TSO1 encodes a

novel nuclear protein required for proper orientation of cell elongation and cytokinesis

during floral organ and integument development (HAUSER et al. 2000; HAUSER et al.

1998; SONG et al. 2000). Severe mutations in the HUELLENLOS (HLL) gene, encoding a

mitochondrial L14 ribosomal subunit, lead to an arrest in ovule growth and degeneration

of the apical regions of the primordia, revealing a role for mitochondrial activity in the

process of ovule growth (SCHNEITZ et al. 1998; SKINNER et al. 2001). Among other

floral effects, reduced activity of the putative protein kinase TOUSLED (TSL) causes

short outer and protruding inner integuments (ROE et al. 1997a; ROE et al. 1997b; ROE et

al. 1993). The variety of protein classes involved in ovule growth implies complex

regulation of this process at the levels of transcription, signal transduction and

metabolism.

SHORT INTEGUMENTS 2 (SIN2) is required for sustaining cell divisions during

integument development (BROADHVEST et al. 2000). At anthesis, sin2-1 ovules have

fewer integument cells than wild type, leaving their nucelli exposed (Figure 1B). sin2-1

also has subtle pleiotropic effects on flower development. The gynoecia of sin2-1

mutants generally have a cleft stigma and occasionally bear an outgrowth on the

corresponding valve. Additionally, in sin2-1 sepals marginal cells appear to be absent or

highly reduced in number. These pleiotropic effects suggest that SIN2 facilitates several

morphogenic pathways.

Double mutants with sin2-1 revealed functional relationships between SIN2 and other

ovule development genes (BROADHVEST et al. 2000). ant-5 sin2-1 double mutants were

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indistinguishable from ant-5 mutants, but ant and sin2 had similar synergistic interactions

with hll. Both ant hll and sin2-1 hll double mutants exhibited a reduced number of

ovules and an earlier abortion of primordia development than hll single mutants. The

ovule effects of sin2-1 were additive with the cell expansion defects of tso1-3. Thus

SIN2 may act downstream of ANT in a common pathway with HLL, and in parallel to

TSO1 to promote cell division during integument growth (BROADHVEST et al. 2000).

In order to better understand regulation of cell division during organ formation we

have identified and characterized the SIN2 gene and isolated a second allele, sin2-2.

SIN2 encodes a putative GTPase of a relatively uncharacterized subclass, termed the

DAR GTPases on the basis of a conserved aspartate-alanine-arginine (DAR) motif and

other conserved features (FU et al. 1998). Some DAR GTPases have been found to be

important for cell division in bacteria, fungi, and human stem cell lines, where they are

associated with assembly or subcellular transport of ribosomal subunits (BASSLER et al.

2001; BIALKOWSKA and KURLANDZKA 2002; KALLSTROM et al. 2003; MATSUO et al.

2006; MORIMOTO et al. 2002; SAVEANU et al. 2001; TSAI and MCKAY 2002; UICKER et

al. 2006). We found SIN2 to localize to mitochondria, and hypothesize a function in

mitochondrial ribosome assembly. In conjunction with hll, sin2 mutants provide an

attractive system with which to study the role of mitochondrial function in the

development of a multicellular organism.

MATERIALS AND METHODS

Plant material: Plants were grown on soil as previously described (KRANZ and

KIRCHHEIM 1987; ROBINSON-BEERS et al. 1992). Some plants were germinated on 1%

agar containing 1% sucrose, 1X Murashige and Skoog salts and 1X Gamborg's B-5

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vitamins (GAMBORG et al. 1968; MURASHIGE and SKOOG 1962). The Students t-test was

used to determine if mutant plants were significantly different from wild type plants in

germination rate, number of days to flowering, number of rosette leaves produced, leaf

production rates, and number of axillary meristems elongation. Plant transformation

vectors and methods were as described (MEISTER et al. 2002).

Genetic mapping and complementation: Using a mapping population generated by

crossing sin2-1 (Landsberg erecta, Ler) with wild type Columbia (Co-3), SIN2 was

previously localized to chromosome 2 between the cleaved amplified polymorphic

sequence (CAPS) m429 and simple sequence length polymorphism (SSLP) AthBIO2

(BROADHVEST et al. 2000). Of 31 chromosomes that had a recombination event between

m429 and AthBIO2, four recombination points were between SIN2 and the SSLP marker

F13H10 Indel2 (Cereon accession CER448978; The Arabidopsis Information Resource

(TAIR) http://www.arabidopsis.org/Cereon/) and one recombination was between SIN2

and a ClaI RFLP within T11A7.16 (At2G41740). This region is spanned by three

overlapping bacterial artificial chromosome (BAC) clones (T11A7, T1K18, and T32G6;

CHOI et al. 1995). Cosmid subclones were generated from BAC T1K18 by partial

digestion with Sau3AI and insertion of 12 – 25 kb fragments in pOCA28-15 (ESHED et al.

1999). The overlapping cosmids cJBT1K18.136, cJBT1K18.105 and CJBT1K18.107

were identified and assembled into a contig using hybridization and fingerprint data.

These cosmids spanned the annotated genes At2G41600.1 to At2G41730.1, and were

transformed into plants from the sin2-1 mapping population. The genotype of transgenic

plants at the SIN2 locus was determined with the F13H10 Indel2 SSLP and the DdeI

CAPS marker 6D20R in At2G42090.1.

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Molecular methods: Coding regions for genes within the complementing cosmid

(cJBT1K18.136) were isolated from sin2-1 homozygous and Ler plants by reverse

transcription polymerase chain reaction (RT-PCR) using gene specific primers and

SuperscriptII reverse transcriptase (Invitrogen, Carlsbad, CA). The cDNAs were

sequenced by Davis Sequencing, Davis CA. Sequences were compared using

Sequencher (Gene Codes Corp., Ann Arbor, MI). Genomic subclones including each of

these genes along with the sequences 5' and 3' extending into the next closest genes were

isolated from cJBT1K18.136 and transformed into plants from the sin2-1 mapping

population. For At2g41670 (SIN2) a 4.0 kb EcoRI fragment was isolated and inserted

into these same sites in pBJ97 (GLEAVE 1992) creating pTH13. At2g42670 was removed

from pTH13 on a NotI fragment and inserted in the same site in pMLBART (GLEAVE

1992) for plant transformation. The 5' end of the SIN2 cDNA was determined by

sequencing clones obtained with the GeneRacer 5'RACE System (Invitrogen, Carlsbad,

CA) using primers specific to SIN2. The full-length cDNA was inserted as an

Spe1/BamHI fragment into XbaI/BamHI digested pMON999 (MEISTER et al. 2002)

creating a P35S::SIN2::NOS3’ transcriptional fusion (pTH52). The cDNA was also

attached to 5.6 kb SIN2 5’-flanking region using a common NruI site in the first exon to

create pTH64. The gene fusions in pTH52 and pTH64 were transferred to pMLBART

(GLEAVE 1992) on NotI fragments for plant transformation.

Primers designed to amplify the SIN2 genomic region, SIN2KOF2 (GAT GGG TTA

TTA CGA TTT GGG CAG TTA TT) and SIN2KOR (CAG TTT CAG GGA CAT CGT

CAA GGA TAA AG), were used to screen the Wisconsin α-population of insertion lines

for additional sin2 alleles as described by Krysan et al. (1999). The insertion site in sin2-

2 was determined by sequencing PCR products using SIN2 primers on either side of the

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insertion and the T-DNA JL202 primer. The SIN2KOR and JL202 primers (KRYSAN et

al. 1999) were used to detect the sin2-2 insertion in segregating populations.

An RT-PCR product generated using synthetic primers T32G6.19F and SIN2GR on

first strand cDNA from Ler inflorescence poly-A+ RNA was cloned into the pCR2.1

vector (Invitrogen, Carlsbad, CA) forming pTH19. The insert was sequenced and found

to include the entire SIN2 coding region with no mutations except for those intentionally

introduced to remove the stop codon. This cDNA was mobilized on an SpeI/BamHI

fragment and inserted into pRJM86 (MEISTER et al. 2002) at the same sites to form a

fusion with the green fluorescent protein (GFP 1.1.15; SCHUMACHER et al. 1999) coding

region. The an SpeI/KpnI fragment containing the SIN2:GFP fusion protein coding

region was inserted between the CaMV 35S promoter and nopaline synthase

polyadenlyation region (NOS3’) in pMON999 (MEISTER et al. 2002). A NotI fragment

consisting of P35S::SIN2:GFP NOS3’ was inserted into the NotI site of the plant

transformation vector pMLBART (GLEAVE 1992) and transformed into plants.

Microscopy: Samples were prepared for scanning electron microscopy and images

were acquired and edited as described (BROADHVEST et al. 2000). Samples were fixed

for light microscopy as described by Baum and Rost (1996). For viewing petal veins,

following fixation, flowers were cleared overnight in a saturated aqueous chlorylhydrate

solution. Petals were removed from the flowers and observed with a Ziess Axioplan

(Ziess Inc., Oberkochen, Germany) microscope using dark field optics. GFP localization

was determined using transgenic SIN2:GFP T2 seedlings which were stained for 1 hr in a

1X MS solution containing the mitochondrial specific stain MitoFluor Red589

(Molecular Probes, Eugene, OR) at 500 nM and were visualized on a Zeiss (Oberkochen,

Germany) Axioscope microscope using fluorescein isothiocyanate and

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tetramethylrhodamine isothiocyanate filter sets respectively. Images were recorded using

the Openlab system (Improvision, Inc., Lexington, MA) and were adjusted for contrast in

Photoshop 6.0 (Adobe Systems, Inc., San Jose, CA).

Phylogenetic analysis: BLAST (ALTSCHUL et al. 1997) searches were used to

identify sequences in GenBank encoding proteins similar to SIN2 and other Arabidopsis

DAR proteins. A large number of proteins containing G motifs were identified. DAR

proteins (defined as those including a DAR-G4-G1-G2-G3 arrangement of motifs but

which did not have two complete G domains in tandem; FU et al. 1998) uniformly gave

the best matches, followed by ENGA proteins. Parsimony analysis was performed with

PAUP4.0b6 (SWOFFORD 1999) on alignments created using ClustalX (THOMPSON et al.

1997). Three separate alignments were produced using gap opening:substitution costs of

2.5:0.5, 5:0.4 and 10:0.3, and the BLOSUM series of weightings (HENIKOFF and

HENIKOFF 1992). ENGA sequences from the ENGA DAR motif to the second G3 motif

were used as an outgroup. Regions unstable between alignments were detected using

SOAP (LOYTYNOJA and MILINKOVITCH 2001). Sequences less than 70% stable were not

included in the phylogenetic analysis (culling). A second analysis was performed in

which all three alignments, arranged in tandem, were included (elision; WHEELER et al.

1995). In parsimony analysis, characters were weighted using a BLOSUM45 amino acid

substitution matrix, which was generated from the published BLOSUM45 matrix

(HENIKOFF and HENIKOFF 1992) by substituting “15 – n” for each value “n” of the matrix.

This results in all positive values, ranging from 0 to 20. An amino acid to gap transition

was given a cost equal to the highest value in the matrix, 20. Bootstrap values were

calculated from 500 bootstrap replicates of heuristic searches on ten random sequence

additions per bootstrap replicate and tree bisection-reconnection procedure for branch

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swapping. Branches that were not consistent between the culling and elision methods of

character weighting or those that had bootstrap values less than or equal to 50% were

collapsed.

Acession numbers: The GenBank/ EMBL accession number for the SIN2 cDNA is

AY254472. The accession numbers for other protein sequences used in the analysis are:

Arabidopsis DGP2 (CAB40031); Arabidopsis DGP3 (AAD15335); Arabidopsis DGP4

(AAF27009); Arabidopsis DGP5 (AAG52287); Arabidopsis DGP6 (AAD26884);

Arabidopsis DGP7 (AAF22888); Oryza DGP4 (BAB61154); Oryza DGP3 (AU077558);

Oryza DGP1 (AAAA01011536); Oryza DGP2 (AC077693); Medicago DGP6

(BG452200); Hordeum DGP5 (BF619769); Zea DGP6 (AAD41267); Lycopersicon

DGP1 (BG133077); Leishmania DGP3 (CAC32254); Leishmania DGP2 (CAC32261);

Leishmania DGP1 (AAF73086); Bacillus ylqF (F69880); Borrelia DGP (AAC67000);

Vibrio DGP (AAF96431); Streptococcus DGP (AAK75264); Synechocystis DGP

(NP_441269); Nostoc DGP (NP_484788); Methanococcus DGP (G64482); Pyrococcus

DGP (F75050); Pyrobaculum DGP (NP_558827); Human MTG1 (AAH04409); Human

LSG1 (NP_060855); Human nucleostemin (GNL3, NP_055181); Human GNL2 (NGP1,

AAH00107); Human HSR1 (XP_041722); Human GNL3L (NP_061940); Drosophila

DGP2 (Dme1_CG3983, AAF55384); Drosophila DGP1 (Dmel_CG6501; AAF56060);

Drosophila DGP4 (Dme1_CG14788, AAF45628); Drosophila DGP5 (Dme1_CG9320,

AAF53920); Drosophila Ngp (AAF57834); Caenorhabditis DGP4 (C53H9.2,

AAK68267); Caenorhabditis DGP3 (T24970); Caenorhabditis nst-1(CAA88860);

Caenorhabditis DGP1 (Y67D2.4, NM_065016); Saccharomyces LSG1 (NP_011416);

Saccharomyces MTG1 (S55083); Saccharomyces NUG1 (S50464); Saccharomyces

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NOG2 (NP_014451); Treponema ENGA (P96128); Aquifex ENGA (O67749); Borrelia

ENGA (O51461); Rickettsia ENGA (NP_360667)

RESULTS

SIN2 encodes a putative GTPase: The ethylmethanesulfonate-induced sin2-1 allele

was previously mapped to chromosome 2 using a sin2-1 Ler / Col. segregating population

(BROADHVEST et al. 2000). Further analysis of this population localized SIN2 to a region

spanned by three overlapping bacterial artificial chromosome (BAC) clones (T11A7,

T1K18, and T32G6:(CHOI et al. 1995)). Figure 2A illustrates the overlapping cosmid

subclones of T1K18 that were transformed into sin2-1 heterozygous progeny from the

mapping population. Eight of the nine identified homozygous sin2-1 plants (genotyped

with mapping markers flanking the SIN2 locus) carrying cJBT1K18.136 exhibited a

wild-type ovule phenotype, indicating complementation of the mutation. cJBT1K18.136

comprises four complete predicted coding regions (At2g41660, 41670, 41680, and

41690; TAIR, http://www.arabidopsis.org/). Subclones spanning these coding regions

from Ler and sin2-1 plants were sequenced and a single gene, At2g41670, was found to

contain a C to T mutation unique to sin2-1 plants. A 4.0 kb Eco RI fragment spanning

At2g41670 (Figure 2B) was also able to complement the sin2-1 mutation, confirming this

gene as SIN2.

RT-PCR generated a 1.25 kb SIN2 cDNA that derived from the eight exons spanning

2 kb of genomic sequence predicted for At2g41670 and encoded a 386 amino acid

protein (Figure 2B). The 5' end of the SIN2 mRNA was determined by 5' RACE PCR to

be thirty-four bases upstream of the putative translation start codon. The complete

coding region of the cDNA was fused to 5.6 kb of genomic sequence 5’ to the SIN2

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coding region, or to the CaMV35S promoter, and both constructs complemented the sin2-

1 mutation. While we were unable to detect SIN2 transcript by in situ hybridization,

transcript was detected by RT-PCR in all structures assayed (Figure 2C). Fusions of

either the 5.6 or 1.5 kb regions 5’ of the SIN2 coding sequence to the GUS coding region

failed to produce GUS activity sufficient to be detected by histochemical staining in

transgenic plants. These observations indicate that SIN2 is likely expressed at low levels

in a variety of plant organs.

The predicted SIN2 protein contains the four sequence motifs (G1-G4) typical of all

members of the GTPase superfamily (BOURNE et al. 1991). The sin2-1 lesion produces a

change of a conserved serine to phenylalanine at position #150 within the P-loop or

putative GTP binding pocket present in the G1 motif (Figure 2B). Because sin2-1 was

the only mutant allele of this gene we had identified, and since mutations in the G1 motif

can have a gain of function effect (BOURNE et al. 1991), it was not possible to assess if

the effects of sin2-1 on plant development were typical of SIN2 loss of function.

Determination of the SIN2 sequence enabled screening for an insertional allele. A

transgenic line in which a T-DNA had inserted in the seventh exon of SIN2, and

associated with deletion of the SIN2 nucleotides corresponding to amino acids 281 to 290

at the insertion site, was identified in the Wisconsin Knock-Out population (KRYSAN et

al. 1999). This lesion was designated sin2-2 (Figure 2B).

Plants heterozygous for sin2-2 were not visibly different from wild-type siblings,

indicating that sin2-2 is a recessive allele. sin2-2 plants produced ovules similar to those

of sin2-1 mutants with a reduced number of ovule integument cells (Figure 1B and 1C;

BROADHVEST et al. 2000). However, the extent of sin2-2 integument reduction was more

variable, with some sin2-2 pistils including as many as six ovules with a nearly wild-type

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appearance in addition to those with short integuments (Figure 1D). These

morphologically wild-type ovules were usually at the distal end of the carpel. Even with

this variation in ovule phenotype, sin2-2 plants were completely female sterile but male

fertile as were sin2-1 plants. The similarity of the most severe integument phenotype

between the sin2-2 insertional mutant and the sin2-1 point mutant suggests that sin2-1 is

a loss of function allele and SIN2 acts to promote cell divisions in the developing

integuments.

sin2-2 illuminates additional developmental roles for SIN2: sin2-1 and sin2-2

plants displayed both overlapping and allele-specific phenotypes. However, it is

possible that some of the specific phenotypic effects observed in sin2-1 and sin2-2 plants

were linked to their respective genetic backgrounds (Ler and Ws respectively). Mutant

phenotypes were always compared to those of wild-type siblings found in the same

segregating population (unless specified otherwise). A reduction in the expected 1:3

(sin2-1:wt) segregation ratio had been previously noted and was proposed to be due to

reduced viability of sin2-1 plants (BROADHVEST et al. 2000). A greater degree of

reduction was observed in sin2-2 plants. When seeds from sin2-2 heterozygote plants

were sown on soil 15% failed to germinate, an additional 12% failed to survive to

maturity and sin2-2 mutants were underrepresented in the population of plants reaching

maturity (3 of 143 total versus 35 of 143 that would be expected). However, the sin2-2

allele was found at the expected frequency among 70 phenotypically wild-type plants (47

of 140 chromosomes, a 1:2.04 ratio of homozygotes to heterozygotes), consistent with

Mendelian transmission of the allele. On media, the sin2-2 germination defect was

partially rescued, with an overall germination efficiency of 98.5 % (336 of 341) in a

segregating population. The under-representation of sin2-2 mutants when grown on soil

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indicates that sin2-2 plants exhibit a greater reduction in viability on soil than sin2-1

plants.

Vegetative growth rates were also affected in sin2 mutants. Both sin2-1 and sin2-2

mutants exhibited an extended germination period. In a sin2-2 segregating population at

day thirteen of growth on media, eighty-two seedlings had only cotyledons while two

hundred fifty-seven had developed visible rosette leaves. Forty-eight of these seedlings

with two leaves >1 mm and forty-eight bearing only cotyledons were transplanted to soil.

All seedlings without leaves that subsequently matured to flowering were shown to be

homozygous for the sin2-2 allele, demonstrating a slower germination rate for sin2-2

seeds compared to wild-type siblings. Detailed analyses showed that both sin2-1 and

sin2-2 seedlings exhibited slower emergence of radicle, cotyledons and secondary leaves

than their wild-type siblings, with sin2-2 showing the strongest effects in regards to

germination and seedling growth (Table 1). The leaf emergence rate of sin2-2 was

reduced by half, but sin2-1 plants behaved as their wild-type siblings (Table 1). The

sin2-2 plants also appeared darker green than their wild type siblings (Supplemental

Figure 1A at http://www.genetics.org/supplemental/). These alterations in germination,

leaf production and greening suggest the involvement of SIN2 activity in a variety of

growth processes during vegetative development.

In addition to the reduction in integument growth, sin2 mutations affected other

aspects of reproductivedevelopment. The number of rosette leaves produced prior to

flowering was increased in both mutants relative to wild type when grown in continuous

light (Table 1). This alteration of flowering time appeared specific to long day conditions

since sin2-1 mutants grown under short day (12 hrs light) produced a wild-type number

of rosette leaves (12.63 + 1.39 vs. 12.85 + 0.96). sin2-2 mutants also showed reduced

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apical dominance, characterized by a doubling in the number of secondary inflorescences,

while sin2-1 did not show any obvious alterations in apical dominance (Table 1). In

some cases, sin2-1 plants had multiple branches originating from the axil of a single

cauline leaf resulting in a bushy phenotype (Supplemental Figure 1 at

http://www.genetics.org/supplemental/). sin2-2 plants also exhibited a marked increase

in vascular discontinuity within the petals relative to wild type, sometimes exhibiting a

completely isolated vein region (Supplemental Figures. 1 and 2 at

http://www.genetics.org/supplemental/). Taken together the pleiotropic effects of both

sin2 mutant alleles suggest SIN2 may be playing a role in the production, perception, or

response to a variety of developmental signals.

SIN2 is a member of a family of atypical GTPases: Database searches indicated

that SIN2 shares several conserved features, including the unique conserved aspartate-

alanine-arginine (DAR) motif, with a relatively uncharacterized family of GTPases, the

DAR GTPases (FU et al. 1998). We performed similarity searches of all publicly

available sequence databases and identified DAR proteins from bacteria, archaea, yeast,

plants and animals. Sequence alignments generated from sixty-one representatives of the

identified proteins revealed additional unreported conserved features. Figure 3 shows

that sequence and order conservation was present within the four G motifs (in the non-

canonical order: G4-G1-G2-G3 (REYNAUD et al. 2005)), the DAR motif and a previously

unreported C-terminal motif. In addition to its unique position, the G4 motif showed the

greatest intersequence variability among the G motifs. However, the typical G4 motif

found in all GTPase superfamily proteins, (Z)4NKXD (were Z is any nonpolar or

hydrophobic amino acid residue and X is any amino acid residue), was found in 80% of

these DAR proteins and seven of the ten deviations from this consensus consisted of

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conservative lysine to arginine or aspartate to glutamate substitutions. The DAR proteins

were also found to invariably have a lysine or arginine one to three amino acids preceding

the four hydrophobic or nonpolar amino acids of the G4 motif. Examination of the DAR

motif region revealed two additional conserved amino acid residues, aspartate (D) and

proline (P), flanking the DAR sequence, and several positions generally consisting of

nonpolar or hydrophobic residues expanding the DAR consensus to D(Z)3XZXDARXP.

The DAR motif was always found N-terminal to the four G motifs in our sample. C-

terminal to the G motifs, a loosely conserved sixth motif, L(X)5G, was also identified.

This additional motif, as well as the DAR motif, is likely to be involved in specific

protein-protein interactions.

While each prokaryotic genome examined included only a single DAR gene, multiple

DAR genes were observed in eukaryotes, with seven members found in Arabidopsis

(SIN2 and DAR GTPases 2–7 (DGP2–7)). Maximum parsimony analysis was used to

evaluate the relationships between fifty-one of the identified DAR proteins selected to

represent the breadth of the entire class. The resulting tree indicated that eukaryotic and

eubacterial DAR proteins fall into four distinct clades, which we have designated 1 – 4 in

Figure 4A. Each clade was found to include representatives from all major groups of

multicellular crown eukaryotes; plants, animals and fungi. Each clade also included at

least one Arabidopsis DGP. This analysis was not sufficient to resolve the relationships

between the archeal proteins and the rest of the DAR proteins or between clades 1–4.

All eubacterial DAR proteins and SIN2 were included in clade 1. Also included in

this clade were the Arabidopsis DGP2 (At4g10650) and DGP3 (At4g02790) proteins as

well as proteins from rice, humans, Drosophila, Caenorhabditis, and Saccharomyces.

Interestingly, some of the non-plant clade 1 proteins were shown to localize to

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mitochondria (BARRIENTOS et al. 2003). SIN2 and DGP2, along with their respective

rice orthologs Oryza SIN2 and Oryza DGP2, appeared to be more closely related to each

other than to any other DAR proteins, suggesting that SIN2 and DGP2 resulted from a

duplication postdating the divergence of plants from other eukaryotes. Thus, it is possible

that these proteins share overlapping functions. DGP3, which includes a predicted

chloroplast transit peptide (data not shown), was placed with high bootstrap support into

a small subgroup within clade 1 containing the cyanobacterial DAR proteins.

Accordingly, DGP3 may play a role in the chloroplast that is analogous to that of SIN2.

DGP4 (At3g07050), DGP5 (At1g52980) and DGP6/7 (At2g27200/At1g08410) were

found in clades 2, 3 and 4 respectively, each of which also includes plant, animal and

fungal representatives, but no bacterial proteins. The clade 4 proteins DGP6 and DGP7

were 72% identical and likely resulted from a relatively recent duplication event. Only

one clade 4 protein was found from each of the other plant species represented in the

sequence databases. Proteins within a particular clade exhibited increased amino acid

conservation among residues flanking each of the six described motifs. Clade 2–4

proteins were longer at both their N- and C-termini than clade 1 proteins. The N-terminal

extensions had conserved features within each group. However, the sequences C-

terminal to the LXG motif were highly variable both among and within groups.

Families of GTPases have been defined by similarities among G1 and G2 sequence

motifs (CALDON et al. 2001). Our database searches and a previous study (REYNAUD et

al. 2005) indicated that the DAR subfamily shares some sequence conservation with

members of the Era family of GTPases, which include Era, EngA, and ThdF/TrmE and

which are found in all eubacteria (CALDON et al. 2001). A comparison of the SIN2 G1

and G2 motifs with the same regions from E. coli Era family members showed that the

- 19 -

G1 sequences GXPNVGKS are conserved between SIN2 and Era family members

(Figure 4B). The secondary structure predicted from the DAR consensus sequence was

compared with the E. coli EngA and Era crystal structures (CHEN et al. 1999; ROBINSON

et al. 2002). All the G motifs of the DAR proteins were predicted to lie within similar

structural regions of EngA and Era (Figure 4D). Within this family, the DAR GTPases

share the greatest sequence similarity with EngA proteins. The G2 sequence PGXT was

found in both SIN2 and EngA (Figure 4B; CALDON et al. 2001) and several EngA

proteins were found of have a D(X)6DAR motif 29 amino acids N-terminal to a G4-G1-

G2-G3 segment (Figure 4C). This is similar to the 22 amino acid distance between the

SIN2 DAR and G4 motifs. In addition, the predicted DAR protein consensus structure

had similar features to the region of EngA from its DAR-like motif to the C-terminal G3

(Figure 4D). These traits suggest SIN2 and other DAR GTPases may be derived from an

EngA-like progenitor.

SIN2 localizes to mitochondria: The Sacchromyces DAR proteins have been found

to localize to different cellular compartments, including the nucleus, cytoplasm and

mitochondria (BARRIENTOS et al. 2003; BASSLER et al. 2001). The sub-cellular

localization of SIN2 was evaluated in transgenic Arabidopsis plants expressing a

SIN2:green fluorescent protein (GFP) fusion protein. The localization of the fused GFP

moiety was determined in the roots, hypocotyls, cotyledons and trichomes of T2

seedlings. Most cells showed a punctate GFP fluorescence pattern that co-localized with

fluorescent stained mitochondria indicative of a mitochondrial localization of the SIN2

protein in planta (Figure 5). This is consistent with the reported localization of those

Sacchromyces and Human DAR proteins that group within the SIN2 clade 1

(BARRIENTOS et al. 2003).

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DISCUSSION

SIN2 is a member of a bacterial/organellar subclass of the DAR GTPase family:

Sequence similarity analyses show that SIN2 includes the DAR motif and reveal a non-

canonical G motif order in the DAR GTPases with the G4 motif preceding G1 through

G3 (Figure 3; FU et al. 1998). Structural similarities were found between the G domains

of DAR proteins and the E. coli Era GTPases, including presence of conserved residues

in the G1 and G2 motifs (Figure 4B), suggesting that the DAR family is closely related to

the Era family (CALDON et al. 2001).

Interestingly, members of the EngA subclass of Era proteins have two canonical G1-

G4 GTPase domains in tandem (Figure 4D; MEHR et al. 2000) and, therefore, contain the

G4-G1-G2-G3 arrangement observed in DAR proteins. In addition, several EngA

proteins include a recognizable D(X)6DAR sequence motif at a position relative to the

first G4 motif that is similar to that seen in DAR proteins (Figure 4C). Notably, there is

additional amino acid conservation between EngA and DAR proteins within the effector-

binding G2 motif (Figure 4B). This level of conservation between EngA and DAR

proteins suggests the two classes derive from a common ancestor that included the

duplication of the canonical G1-G4 GTPase domains and a DAR motif, and that the DAR

proteins derived from this common ancestor through truncation (Figure 4B-4D).

Eukaryotic DAR proteins resolve into four moderately to well supported clades (1-4),

suggesting a early expansion of the gene family (Figure 4A; REYNAUD et al. 2005). In

contrast, eubacterial DAR GTPases are confined to clade 1, along with SIN2, DPG2,

DGP3 and other eukaryotic representatives. This association is supported by another

recent phylogenetic analysis (REYNAUD et al. 2005). These results suggest that the

- 21 -

eukaryotic proteins in this clade could have been acquired in an early endosymbiotic

event or other horizontal gene transfer. The first possibility is consistent with the

mitochondrial localization of SIN2 (this work) and of its putative orthologs in yeast and

mammals (BARRIENTOS et al. 2003). The sequence similarity and close association

between SIN2, DGP2 and plant orthologs in the phylogram indicates that DGP2 is also

likely a mitochondrial protein that derived from a recent gene duplication event. In

contrast, DGP3 and a rice ortholog associate with the DAR proteins from known bacterial

relatives of chloroplasts (Figure 4) and include putative chloroplast transit peptides (data

not shown). Thus, DGP3 likely represents the chloroplast DAR ortholog in higher plants,

as has been previously hypothesized (REYNAUD et al. 2005). The structure of clade 1

therefore suggests that each of the two endosymbiotic events related to plant evolution,

acquisition of mitochondria and plastids, brought with them specific DAR GTPase

lineages.

DAR proteins are important for ribosomal assembly in many subcellular

compartments: Recent reports shed light on the biological function of some of the clade

1 proteins that are putative orthologs of SIN2. B. subtilis YlqF (Figure 4A) was shown to

facilitate an essential step in assembly of the large ribosomal subunit (MATSUO et al.

2006; UICKER et al. 2006). Cells deficient in YlqF accumulate incomplete 50S ribosomal

subunits lacking the L16 and L25 protein components (MATSUO et al. 2006; UICKER et

al. 2006). Evidence of participation of the GTPase activity of YlqF in the assembly

process is provided by the stimulation of YlqF GTPase activity in the presence of 50S

subunits, and an association of YlqF with 50S subunits upon addition of a non-

hydrolysable GTP analog (MATSUO et al. 2006). Functional studies on humans and

Saccharomyces MTG1 proteins indicate conservation of the ribosome assembly function

- 22 -

for these proteins in the eubacterial-derived mitochondria. Saccharomyces MTG1 was

shown to be important for translation in mitochondria and a role in assembly of the

mitochondrial large ribosomal subunit was hypothesized (BARRIENTOS et al. 2003). It

was further shown that the human MTG1 protein could partially complement the

Saccharomyces mtg1 mutant, indicating that the ribosome biogenesis function is likely

also conserved in animals (BARRIENTOS et al. 2003).

Other diverse DAR proteins have also been found to participate in ribosome

biogenesis in other sub cellular compartments. The Saccharomyces clade 2 protein

NUG1 appears to be involved in ribosome assembly in the nucleus and transport through

nuclear pores (BASSLER et al. 2001; NISSAN et al. 2002). The Saccharomyces clade 3

protein NOG2 has functions similar to NUG1 and in addition may be involved in mRNA

processing (BASSLER et al. 2001; BIALKOWSKA and KURLANDZKA 2002; SAVEANU et al.

2001). LSG1, the Saccharomyces clade 4 protein, is located in the cytoplasm and is also

important for ribosome transport through nuclear pores (KALLSTROM et al. 2003; NISSAN

et al. 2002). This indicates conservation of a ribosome biogenesis function among

divergent DAR GTPase in the subcellular compartments where they reside.

The reported functions of SIN2 orthologs as well as the mitochondrial localization of

SIN2 and of its closest eukaryotic orthologs suggests strongly that SIN2 is a DAR

GTPase that functions in mitochondrial ribosome assembly in Arabidopsis. SIN2 mRNA

was present in all tested plant parts as would be expected for a mitochondrial protein.

Effects of SIN2 reduced activity in Arabidopsis are consistent with a role in

supporting mitochondrial translation: At the molecular level, the mutation found in the

sin2-1 allele would generate a protein with a single amino acid substitution in the GTP

loop, the GTP-binding motif of the GTPase. Mutations in this motif were identified in

- 23 -

other GTPases and led to gain or loss of function phenotypes (BOURNE et al. 1991).

However, the phenotypic overlap observed between the sin2-1 and the insertional sin2-2

allele suggests that both alleles cause loss of function or a decrease in SIN2 protein

activity.

Mitochondria are involved in biochemical energy utilization and one would expect

lower energy levels to translate into reduced or retarded growth. The reduced growth

observed in the integuments of sin2 ovules resulted from a decrease in the number of cell

divisions in these structures (BROADHVEST et al. 2000, this study). This reduced growth

could result from insufficient energy production due to compromised mitochondrial

activity. Indeed, we have previously characterized the phenotypic effects of mutations in

HLL, which encodes a mitochondrial ribosomal protein homologous to the L14 proteins

of Eubacteria (SKINNER et al. 2001). hll mutations result in a dramatic reduction in ovule

growth, apparently due to compromised translation in mitochondria (SCHNEITZ 1999;

SKINNER et al. 2001). The combination of sin2-1 and hll-1 mutations is synergistic,

leading to almost complete loss of ovule primordial growth (BROADHVEST et al. 2000).

This could be due to the combination of effects of the two mutations leading to even

more severe deficit in mitochondrial function, at least during ovule development.

Interestingly, the ant mutation was found to be epistatic to sin2-1 but synergistic with hll

leading to a similar but somewhat less severe phenotype in comparison to the sin2-1 hll-1

double mutant (BROADHVEST et al. 2000). This supports a hypothesis that ANT is

needed to promote growth and HLL is needed for growth to actually occur. SIN2 would

then facilitate the role of HLL in ovule and integument growth. Thus, this suggests that

HLL is more important for mitochondrial activity than SIN2, at least during ovule

- 24 -

development, consistent with the proposed structural and assembly roles for the two

proteins with respect to the large ribosomal subunit.

Many of the other phenotypic effects observed for sin2 Arabidopsis plants such as

slower growth, reduced pollen formation and reduced organ size, are consistent with a

general decrease in energy metabolism that would be expected for plants with reduced

mitochondrial activity. However, more complex phenotypes observed also suggest a role

for mitochondria in integration of signaling pathways. For example, maintenance of

axillary bud dormancy and continuity of vascular tissue are both under control of auxin-

related pathways (CHATFIELD et al. 2000; HARDTKE and BERLETH 1998). In addition,

inhibition of auxin transport or mutation of the auxin response factor gene ETTIN causes

differential effects along the length of the gynoecium similar to the gradation of effects

observed for sin2-2 (NEMHAUSER et al. 2000). Thus, some aspects of SIN2 action may

reflect an interaction between phytohormone signaling and mitochondrial function. The

broad range of effects that sin2 mutations have on plant growth and architecture is

consistent with the SIN2 GTPase regulating mitochondrial activity in a variety of

developmental pathways within the plant.

The observation that available sin2 mutants are not more seriously growth deficient

suggests that significant mitochondrial function likely persists in these mutant plants.

This implies either that SIN2 activity is not always required for assembly of the

mitochondrial translational apparatus, or that a redundant activity exists. Such a

redundant activity could be supplied by DGP2, which our sequence and phylogenic

studies have identified as a late-branching paralog of SIN2. DGP2 could have

overlapping function with SIN2 and could partially compensate for the reduction or loss

of SIN2 activity in the sin2 mutants. Preliminary analyses of Arabidopsis dgp2 mutants

- 25 -

show that this gene is functional and seem to affect whole plant architecture (Theresa

Hill, unpublished results). This would parallel the observed redundancy for HLL and the

paralogous HULLENLOS PARALOG (HLP) gene (SKINNER et al. 2001). More studies

will be needed to evaluate the role of DGP2 in mitochondria and putative paralogous

interactions with SIN2.

Complexity in mitochondrial modulation of growth pathways: In plants,

mitochondria have been shown to be important for male fertility, appropriate expression

of floral homeotic genes, and programmed cell death (CHRISTENSEN et al. 2002;

HOEBERICHTS and WOLTERING 2003; LEINO et al. 2003; LINKE et al. 2003). Work in our

laboratory has shown that at least two ribosomal proteins targeted to the mitochondria,

HLL and SIN2, are necessary for ovule development and both also affect regulation of

other developmental pathways in Arabidopsis. Interestingly, both of these genes have

functional paralogs in Arabidopsis, DGP2, and HLP, respectively. Together regulation of

these genes could allow for modulation of mitochondrial activity to facilitate differential

growth necessary for morphogenesis. The added complexity that cell mitochondrial

number can vary in a tissue and developmental dependant manner provide additional

plasticity for mitochondria integrated pathways. SIN2 and HLL, along with their

paralogs, offer an entry point to understand the regulation and impact of mitochondrial

protein synthesis on plant development.

- 26 -

ACKNOWLEDMENTS

The authors would like to thank Chris Roxas and Roderick Kumimoto for dedicated

assistance in plant care and genotyping, Yuval Eshed for the gift of pOCA28-15, the

National Science Foundation Arabidopsis Biological Resource Center at Ohio State for

Arabidopsis DNA clones, and members of the Gasser and Bowman Labs for helpful

discussions. This work was supported by National Science Foundation Grant IBN-

0079434 and by a United States Department of Agriculture National Research Initiative

Competitive Grants Program award 2001-35304-09989.

- 27 -

LITTERATURE CITED

ALTSCHUL, S. F., T. L. MADDEN, A. A. SCHAFFER, J. ZHANG, Z. ZHANG et al., 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.

BAKER, S. C., K. ROBINSON-BEERS, J. M. VILLANUEVA, J. C. GAISER and C. S. GASSER, 1997 Interactions among genes regulating ovule development in Arabidopsis thaliana. Genetics 145: 1109-1124.

BARRIENTOS, A., D. KORR, K. J. BARWELL, C. SJULSEN, C. D. GAJEWSKI et al., 2003 MTG1 codes for a conserved protein required for mitochondrial translation. Mol. Biol. Cell 14: 2292-2302.

BASSLER, J., P. GRANDI, O. GADAL, T. LESSMANN, E. PETFALSKI et al., 2001 Identification of a 60S preribosomal particle that is closely linked to nuclear export. Mol. Cell 8: 517-529.

BAUM, S. F., and T. L. ROST, 1996 Root apical organization in Arabidopsis thaliana 1. Root cap and protoderm. Protoplasma 192: 178-188.

BIALKOWSKA, A., and A. KURLANDZKA, 2002 Additional copies of the NOG2 and IST2 genes suppress the deficiency of cohesin Irr1p/Scc3p in Saccharomyces cerevisiae. Acta Biochim. Pol. 49: 421-425.

BOURNE, H. R., D. A. SANDERS and F. MCCORMICK, 1991 The GTPase superfamily: conserved structure and molecular mechanism. Nature 349: 117-127.

BROADHVEST, J., S. C. BAKER and C. S. GASSER, 2000 SHORT INTEGUMENTS 2 promotes growth during Arabidopsis reproductive development. Genetics 155: 895-907.

CALDON, C. E., P. YOONG and P. E. MARCH, 2001 Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function. Mol. Microbiol. 41: 289-297.

CHATFIELD, S. P., P. STIRNBERG, B. G. FORDE and O. LEYSER, 2000 The hormonal regulation of axillary bud growth in Arabidopsis. Plant J. 24: 159-169.

CHEN, X., D. L. COURT and X. H. JI, 1999 Crystal structure of ERA: A GTPase-dependent cell cycle regulator containing an RNA binding motif. Proc. Nat. Acad. Sci. USA 96: 8396-8401.

CHOI, S., R. A. CREELMAN, J. E. MULLET and R. A. WING, 1995 Construction and characterization of a bacterial artificial chromosome library of Arabidopsis thaliana. Weeds World 2: 17-20.

CHRISTENSEN, C. A., S. W. GORSICH, R. H. BROWN, L. G. JONES, J. BROWN et al., 2002 Mitochondrial GFA2 is required for synergid cell death in Arabidopsis. Plant Cell 14: 2215-2232.

CUFF, J. A., M. E. CLAMP, A. S. SIDDIQUI, M. FINLAY and G. J. BARTON, 1998 JPred: a consensus secondary structure prediction server. Bioinformatics 14: 892-893.

ELLIOTT, R. C., A. S. BETZNER, E. HUTTNER, M. P. OAKES, W. Q. J. TUCKER et al., 1996 AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. Plant Cell 8: 155-168.

ESHED, Y., S. F. BAUM and J. L. BOWMAN, 1999 Distinct mechanisms promote polarity establishment in carpels of Arabidopsis. Cell 99: 199-209.

FU, G., S. MELVILLE, S. BREWSTER, J. WARNER and D. C. BARKER, 1998 Analysis of the genomic organisation of a small chromosome of Leishmania braziliensis M2903

- 28 -

reveals two genes encoding GTP-binding proteins, one of which belongs to a new G-protein family and is an antigen. Gene 210: 325-333.

GAISER, J. C., K. ROBINSON-BEERS and C. S. GASSER, 1995 The Arabidopsis SUPERMAN gene mediates asymmetric growth of the outer integument of ovules. Plant Cell 7: 333-345.

GAMBORG, O. L., R. A. MILLER and K. OJIMA, 1968 Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158.

GLEAVE, A. P., 1992 A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20: 1203-1207.

HARDTKE, C. S., and T. BERLETH, 1998 The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO J. 17: 1405-1411.

HAUSER, B. A., J. HE, S. O. PARK and C. S. GASSER, 2000 TSO1 is a novel protein regulating cell division and directional cell expansion in Arabidopsis. Development 127: 2219-2226.

HAUSER, B. A., J. M. VILLANUEVA and C. S. GASSER, 1998 Arabidopsis TSO1 regulates directional processes in cells during floral organogenesis. Genetics 150: 411-423.

HENIKOFF, S., and J. G. HENIKOFF, 1992 Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 89: 10915-10919.

HOEBERICHTS, F. A., and E. J. WOLTERING, 2003 Multiple mediators of plant programmed cell death: interplay of conserved cell death mechanisms and plant-specific regulators. Bioessays 25: 47-57.

KALLSTROM, G., J. HEDGES and A. JOHNSON, 2003 The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively. Mol. Cell. Biol. 23: 4344-4355.

KLUCHER, K. M., H. CHOW, L. REISER and R. L. FISCHER, 1996 The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2. Plant Cell 8: 137-153.

KRANZ, A. R., and B. KIRCHHEIM, 1987 Handling of Arabidopsis, pp. 4.1.1-4.2.7 in Arabidopsis Information Service, v. 24: Genetic Resources in Arabidopsis, edited by A. R. KRANZ. Arabidopsis Information Service, Frankfurt, Germany.

KRYSAN, P. J., J. C. YOUNG and M. R. SUSSMAN, 1999 T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11: 2283-2290.

LEINO, M., R. TEIXEIRA, M. LANDGREN and K. GLIMELIUS, 2003 Brassica napus lines with rearranged Arabidopsis mitochondria display CMS and a range of developmental aberrations. Theor. Appl. Genet. 106: 1156-1163.

LINKE, B., T. NOTHNAGEL and T. BORNER, 2003 Flower development in carrot CMS plants: mitochondria affect the expression of MADS box genes homologous to GLOBOSA and DEFICIENS. Plant J. 34: 27-37.

LOYTYNOJA, A., and M. C. MILINKOVITCH, 2001 SOAP, cleaning multiple alignments from unstable blocks. Bioinformatics 17: 573-574.

MATSUO, Y., T. MORIMOTO, M. KUWANO, P. C. LOH, T. OSHIMA et al., 2006 The GTP-binding protein, YlqF, participates in the late step of 50S ribosomal subunit assembly in Bacillus subtilis. J. Biol. Chem. M512556200.

- 29 -

MEHR, I. J., C. D. LONG, C. D. SERKIN and H. S. SEIFERT, 2000 A homologue of the recombination-dependent growth gene, rdgC, is involved in gonococcal pilin antigenic variation. Genetics 154: 523-532.

MEISTER, R. J., L. M. KOTOW and C. S. GASSER, 2002 SUPERMAN attenuates positive INNER NO OUTER autoregulation to maintain polar development of Arabidopsis ovule outer integuments. Development 129: 4281-4289.

MORIMOTO, T., P. C. LOH, T. HIRAI, K. ASAI, K. KOBAYASHI et al., 2002 Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis. Microbiology-Uk 148: 3539-3552.

MURASHIGE, T., and F. SKOOG, 1962 A revised medium for rapid growth and bioassays with tobaco tissue cultures. Physiol. Plant. 15: 473-479.

NEMHAUSER, J. L., L. J. FELDMAN and P. C. ZAMBRYSKI, 2000 Auxin and ETTIN in Arabidopsis gynoecium morphogenesis. Development 127: 3877-3888.

NISSAN, T. A., J. BASSLER, E. PETFALSKI, D. TOLLERVEY and E. HURT, 2002 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm. EMBO. J. 21: 5539-5547.

REYNAUD, E. G., M. A. ANDRADE, F. BONNEAU, T. B. LY, M. KNOP et al., 2005 Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization. BMC Biol. 3: 21.

ROBINSON, V. L., J. HWANG, E. FOX, M. INOUYE and A. M. STOCK, 2002 Domain Arrangement of Der, a Switch Protein Containing Two GTPase Domains. Structure 10: 1649-1658.

ROBINSON-BEERS, K., R. E. PRUITT and C. S. GASSER, 1992 Ovule development in wild-type Arabidopsis and two female-sterile mutants. Plant Cell 4: 1237-1249.

ROE, J. L., T. DURFEE, J. R. ZUPAN, P. P. REPETTI, B. G. MCLEAN et al., 1997a TOUSLED is a nuclear serine-threonine protein kinase that requires a coiled-coil region for oligomerization and catalytic activity. J. Biol. Chem. 272: 5838-5845.

ROE, J. L., J. L. NEMHAUSER and P. C. ZAMBRYSKI, 1997b TOUSLED participates in apical tissue formation during gynoecium development in Arabidopsis. Plant Cell 9: 335-353.

ROE, J. L., C. J. RIVIN, R. A. SESSIONS, K. A. FELDMANN and P. C. ZAMBRYSKI, 1993 The TOUSLED gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development. Cell 75: 939-950.

SAVEANU, C., D. BIENVENU, A. NAMANE, P. E. GLEIZES, N. GAS et al., 2001 Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps. EMBO J. 20: 6475-6484.

SCHNEITZ, K., 1999 The molecular and genetic control of ovule development. Curr. Opin. Plant Biol. 2: 13-17.

SCHNEITZ, K., S. C. BAKER, C. S. GASSER and A. REDWEIK, 1998 Pattern formation and growth during floral organogenesis: HUELLENLOS and AINTEGUMENTA are required for the formation of the proximal region of the ovule primordium in Arabidopsis thaliana. Development 125: 2555-2563.

SCHNEITZ, K., M. HULSKAMP and R. E. PRUITT, 1995 Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. Plant J. 7: 731-749.

- 30 -

SCHUMACHER, K., D. VAFEADOS, M. MCCARTHY, H. SZE, T. WILKINS et al., 1999 The Arabidopsis det3 mutant reveals a central role for the vacuolar H(+)-ATPase in plant growth and development. Genes Dev. 13: 3259-3270.

SKINNER, D. J., S. C. BAKER, R. J. MEISTER, J. BROADHVEST, K. SCHNEITZ et al., 2001 The Arabidopsis HUELLENLOS gene, which is essential for normal ovule development, encodes a mitochondrial ribosomal protein. Plant Cell 13: 2719-2730.

SKINNER, D. J., T. A. HILL and C. S. GASSER, 2004 Regulation of ovule development. Plant Cell 16: S32-45.

SONG, J.-Y., T. LEUNG, L. K. EHLER, C. WANG and Z. LIU, 2000 Regulation of meristem organization and cell division by TSO1, an Arabidopsis gene with cysteine-rich repeats. Development 127: 2207-2217.

SWOFFORD, D. L., 1999 PAUP*: phylogenetic analysis using parsominy (and other methods), version 4.0. Sinauer Associates, Sunderland, Massachusetts.

THOMPSON, J. D., T. J. GIBSON, F. PLEWNIAK, F. JEANMOUGIN and D. G. HIGGINS, 1997 The CLUSTAL*X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882.

TSAI, R. Y., and R. D. MCKAY, 2002 A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev. 16: 2991-3003.

UICKER, W. C., L. SCHAEFER and R. A. BRITTON, 2006 The essential GTPase RbgA (YlqF) is required for 50S ribosome assembly in Bacillus subtilis. Mol. Microbiol. 59: 528-540.

VILLANUEVA, J. M., J. BROADHVEST, B. A. HAUSER, R. J. MEISTER, K. SCHNEITZ et al., 1999 INNER NO OUTER regulates abaxial-adaxial patterning in Arabidopsis ovules. Genes Dev. 13: 3160-3169.

WHEELER, W., C., J. GATESY and R. DESALLE, 1995 Elision: A method for accommodating multiple molecular sequence alignments with alignment-ambiguous sites. Mol. Phylo. Evol. 4: 1-9.

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FIGURE LEGENDS

FIGURE 1. Scanning electron micrographs of stage 3-VI ovules (anthesis, stages from

Schneitz et al., (1995)). (A) Wild-type Ler; (B) sin2-1; (C) and (D) sin2-2 plants. In

(D) the gradation in severity of effects of sin2-2 from the base to the apex of the carpel is

apparent. f, funiculus; ii, inner integument; n, nucellus; oi, outer integument. Bars are 50

µm (A-C) and 100 µm (D).

FIGURE 2. Identification and expression of the SIN2 gene. (A) The chromosomal region

surrounding the SIN2 locus with the molecular markers used to map SIN2 shown above,

and the number of recombination events between markers found in 918 F2 plants

indicated below. The BAC T1K18 spans part of the region between the closest flanking

markers. Cosmid subclones .105, .136 and .107 derived from T1K18 and used in the

complementation test are illustrated. (B) The 4.0 kb EcoRI fragment spanning a single

gene, At2G41670, derived from cJBT1K18.136, that was able to complement the sin2-1

mutant phenotype. The exons in this region and the transcriptional start site as

determined by RT PCR are shown as boxes and a bent arrow respectively. Arrows

indicate the positions of primers used to screen T-DNA lines for additional sin2 alleles.

The position of the missense (sin2-1) and insertional (sin2-2) mutations are indicated.

Numbering is relative to the translational start site. (C) RT-PCR with total RNA from

several plant parts used as template shows that SIN2 RNA was present in all structures

assayed.

- 32 -

FIGURE 3. DAR protein alignment. An alignment of the seven Arabidopsis DAR

proteins, SIN2 and DGP2-7, and the human SIN2 ortholog, Human DGP1, highlights the

six conserved motifs of this family. Residues found in all of these proteins are shown in

white on black. These include amino acids within the GTPase motifs, G1-G4, and the

DAR motif, labeled and underscored below each sequence. There is also a region C-

terminal to the recognizable GTPase motifs which appears loosely conserved and

includes a L(X)5G motif. Amino acids conserved between SIN2 and the closely related

proteins DGP2 (38% identity, 57% similarity) and Human DGP1 (31% identity, 47%

similarity) are highlighted in grey. Residues conserved between SIN2 and DGP2 or

HsDGP1 are shown in black or white letters respectively. The positions corresponding to

the sin2-1 S150F transition and the sin2-2 insertion are indicated above the sequence.

FIGURE 4. Phylogenetic analysis of DAR GTPases. (A) The single tree generated by

parsimony analysis of a culled alignment was modified to collapse branches that were not

supported in both culling and elision analyses by bootstrap values greater that 50%. Four

main clades are resolved, designated clades 1-4. Eukaryotes are represented in each clade.

Prokaryotic sequences are found in clade 1 and two orphan branches (including

Methanococcus and Pyrobaculus sequences). Arabidopsis proteins are highlighted with

grey boxes with SIN2 outlined in black. Numbers on the branches indicate bootstrap

values based on 500 bootstrap replicates. Branch lengths are relative to distance as

calculated using the transformed BLOSUM45 matrix. Clade designations are shown

above the respective branches. Proteins without previous names were given “DGP”

designations. (B) to (D) DAR proteins share conserved sequences and secondary

- 33 -

structure with Era family members. (B) An alignment of the G1 and G2 motifs of SIN2,

E. coli Era, ThdF, EngA1 and EngA2 demonstrates that the SIN2 G1 motif is similar to

that of Era family members (Era, ThdF, and EngA). (C) A region of EngA similar to a

DAR motif was found between EngA1 G3 and EngA1 G4. (D) A comparison of the

clade 1 consensus secondary structure predicted by the Jpred server (CUFF et al. 1998)

with that derived from the E. coli EngA and E. coli Era crystal structures (CHEN et al.

1999; ROBINSON et al. 2002). EngA structure (arrorws and cylinders indicate ß-sheet and

α-helix regions, respectively) appears to derive from a direct repeat of two Era-like units.

The DAR GTPases, represented by SIN2, resemble the central (G4 to G3) region of those

EngA proteins that include DAR motifs.

FIGURE 5. Localization of SIN2:GFP in Arabidopsis. Black and white images of

Arabidopsis hypocotyl cells transformed with 35S:SIN2:GFP. Fluorescence appears

white. (A) Green fluorescent protein is localized to small particles within the cells which

correspond to mitochondria stained with MitoFluor 589 dye shown in B.

- 34 -

TABLE 1

Growth measurements

Days to

Radicle

Emergence

Days to

Cotyledon

emergence

Days to

Two Leaves

Leaf Emergence

(days/leaf)

Days to Bolting Number of

Rosette Leaves

Number of

Active Branches

SIN2

(Ler)

2.2 + 1.2

(57)

4.7 + 1.5

(57)

10.7 + 0.8

(57)

2.8

R2 = 0.98

29.6 + 3.4 (57) 5.4 + 1.0 (57) 3.0 + 0.6 (17)

sin2-1

(Ler)

2.6 + 0.7

(10)

7.2 + 2.3

(10)**

12.2 + 2.2

(10)

2.2

R2 = 0.99

34.6 + 1.9

(14)***

7.6 + 1.4

(14)***

3.2 + 0.7

(12)

SIN2

(Ws)

1.1 + 0.2

(39)

3.1 + 0.6

(39)

9.1 + 0.4

(39)

2.2

R2 = 0.98

24.5 + 1.7 (36) 6.1 + 0.9

(36)

4.1 + 0.7

(16)

sin2-2

(Ws)

3.7 + 1.5

(23)***

9.1 + 1.8

(23)***

19.2 + 5.5

(23) ***

5.1

R2 = 0.98

49.4 + 9.2

(19)***

7.6 + 2.3 (13)^ 10.4 + 2.4

(11)***

Numbers represent the mean + the standard deviation of the mean. Statistically significant differences between wt-1 and sin2-1 or wt-2

and sin2-2 are designated ^ p < 0.05, * p < 0.01, ** p < 0.001, *** p < 0.0001 where p values are determined by the students T-test.

EcoRI(-1813)

sin2-1S150 >F

sin2-2Δ aa 280-291

EcoRI(+2217)

B

m429 BIO2F13H10Indel2

SIN2T11A7.16

18 1 8

T1K18

.136.105 .107

Figure 2

A

4

C

Figure 3.

DGP3 MFAQ-----------------LAPSP------------SPSPANVSHFVHRRTFRQCTYAVSAS--------------------------------LPTANSSRPPPIQANIIVGGKDLNLDVTRKDDSIG----------FKLEANE------- 77 DGP4 MVKRSK---------------KSKSK----------RVTLKQKHKVLKKVKEHHKKKAKDAKKLG----------------------LHRKPRVEKDPGIPNDWPFKEQELKALEVRRARALEEIEQKKEARKE--------RAKKRK------- 93 DGP5 MVKKEKKANVSGKPKHSLDANRADGKKKTTETRSKSTVNRLKMYKTRPKR-NAGGKILSNEYQSKELPNSRIAPDRRWFGNTRVVNQKELEYFREELQTKMSSNYNVILKERKLPMSLLTDNKKQSRVHLLDMEPFQDAFGRKTKRKRPKLVASD 154 DGP6 MGKNEK---------------TSLGR---------ALVKHHNHMIQETKEKGKSYKDQHKKVLESVTEVSDID------------AIIEQAEEAERLFAIHHDSATPVPINMDTGSSSSGITAKEWKEQRMREEALHASS-LQVPRRPHWTPKMN 118 DGP7 MGKSEK---------------TSLGR---------SLVKHHNHMIQESKDKGKYYKNLQKKVLESVTEVSDID------------AIIEQAEEAERLYTINHSSSTPLSINLDTNSSSSVIAAEEWREQQKIEEALHASS-LQVPRRPPWTPEMS 118

SIN2 ---------------MVMMLKKTVKKGLIGGMSFAKDAGKINWFPGHMAAATRAIRNRLKLSDLVIEVRDARIPLSS-ANE-DLQ--SQMS-AKRRIIALNKKDLANPNVLNKWTRHFESS--------------------------------KQ 103 DGP2 ---------------MATAKTWKIAREIGDAVIKASRNPNRRWYGPHMAAAVRAISERIPLVDFVLEIRDARIPLSS-EYE-LLRKFSPLP-SKR-IIVLNKMELADPLELKKCIDYFEERN--------------------------------Y 104HsDAR1 -------------MRLTPRALCSAAQAAWRENFPLCGRDVARWFPGHMAKGLKKMQSSLKLVDCIIEVHDARIPLSG-RNPLFQETLG----LKPHLLVLNKMDLADLTEQQKIMQHLEGEG-------------------------------LK 127 DGP3 -DEIDWMNLESD------------------IRLWTRALRPVQWYPGHIMKTEKELREQLKLMDVVIEVRDARIPLST-THP---KMDAWLG-NRKRILVLNREDMISNDDRNDWARYFAKQG--------------------------------I 176 DGP4 LGLVDDEDTKTEG------------------ETIEDLPKVVNVRDNSERAFYKELVKVIELSDVILEVLDARDPLGTRCTDMERMVMQAGP-NKHLVLLLNKIDLVPREAAEKWLMYLREEFP-------------------------AVAFKCS 204 DGP5 YEALVKKAAESQDAFEEKNGAGPSGEGGEEEDGFRDLVRHTMFEKGQSKRIWGELYKVIDSSDVIVQVIDARDPQGTRCHHLEKTLKEHHK-HKHMILLLNKCDLVPAWATKGWLRVLSKEY--------------------------------P 276 DGP6 VEKLDANEKQAFLTWRRK-----------LASLEEN-EKLVLTPFEKNLDIWRQLWRVLERSDLIVMVVDARDPLFYRCPDLEAYAQEID-EHKKTMLLVNKADLLPSYVREKWAEYFSRNNILFVFWSAKAATATLEGKPLKEQWRAPDTTQKT 260 DGP7 VEELDANEKQAFLNWRRM-----------LVSLEEN-EKLVLTPFEKNLDIWRQLWRVLERSDLIVMVVDARDPLFYRCPDLEAYAQEID-EHKKIMLLVNKADLLPTDVREKWAEYFRLNNILFVFWSAIAATATLEGKVLKEQWRQPDNLQKT 260 DAR G4____

sin2-1 S>F SIN2 DCIAINAHSRSSVMKLLDLVELKLKEV-IAR---------------EPTLLVMVVGVPNVGKSALINSIHQIAAARFPVQERLKRATVGPLPGVTQDIAGFKIAH-RPSIYVLDSPGVLVPSIPDIETGLKLALSGSVKDSVVGEERIAQYFLAI 241 DGP2 LSYAVNSHNKDCVKQLLNFLQSQVRELHKAG-------------HSGHTTTMMLLGIPNVGKSALSNSLHHIGRISAAEKGKLKHTTVSSQPGDTKDIMSLKIGS-HPNVYVLDTPGIFPPNLYDAEICAKLALTGAIPDDIVGELKLARLFLTI 245HsDAR1 NVIFTNCVKDENVKQIIPMVTELIGRSHRYHR------------KENLEYCIMVIGVPNVGKSSLINSLRRQHLRKG------KATRVGGEPGITRAVMSKIQVSERPLMFLLDTPGVLAPRIESVETGLKLALCGTVLDHLVGEETMADYLLYT 264 DGP3 KVIFTNGKLGMGAMKLGRLAKSLAGDVNGKRREKG---------LLPRSVRAGIIGYPNVGKSSLINRLLK-----------RKICAAAPRPGVTREMK---WVKLGKDLDLLDSPGMLPMRIDDQAAAIKLAICDDIGEKAYDFTDVAGILVQM 308 DGP4 TQEQRSNLGWKSSKASKPSNMLQTSDCLGADTLIKLLKNYSRSHELKKSITVGIIGLPNVGKSSLINSLKR-----------AHVVNVGATPGLTRSLQ---EVHLDKNVKLLDCPGVVMLKSSG-NDASIALRNCKRIEKLDDPVSPVKEILK- 343 DGP5 TLAFHASVNKSFGKGSLLSVLRQFARLKSDK----------------QAISVGFVGYPNVGKSSVINTLRT-----------KNVCKVAPIPGETKVWQ---YITLTKRIFLIDCPGVVYQS--RDTETDIVLKGVVRVTNLE---DASEHIGEV 396 DGP6 DNPAVKVYGRDDLLDRLKLEALEIVKMRKSRGVSATS-----TESHCEQVVVGFVGYPNVGKSSTINALVG-----------QKRTGVTSTPGKTKHFQTLIISED---LMLCDCPGLVFPSFSSSRYEMVASGVLPIDRMTEHLEAIKVVAELV 396 DGP7 DDPDIMIYGRDELLSRLQFEAQEIVKVRNSRAASVSSQ-SWTGEYQRDQAVVGFVGYPNVGKSSTINALVG-----------QKRTGVTSTPGKTKHFQTLIISDE---LMLCDCPGLVFPSFSSSRYEMIASGVLPIDRMTEHREAIQVVADKV 400 G1 G2 G3__

| sin2-2 | SIN2 LNIRGTPLHWKYLVEGINEGPHADCIDKPSYNLKDLRHQRTKQPDSSALHYVGDMISEVQRSLYITLSEFDGDTEDENDLECLIEQQFEVLQKALKIPHK-ASEARLMVSKKFLTLFRTGRLGPFILDDVPETETDHPNSKRVVVL--------- 386 DGP2 LNSSHEYKKWAKLCKSQDLTESLSDESSKS--------DAKHKRQYATDHTQDFIVYDVRRVLYETISAFDGNLEDEISMGNLIETQFAALRSVLRVPEEASEFADLRVASKILNLYRTGRLGHYTLEHVSALAKSYTYL--------------- 377HsDAR1 LNKHQRFGYVQHYG--------------------------------------------------LGS-ACDNVERVLKSVAVKLGKTQKVKVLTGTGNVNVIQPNYPAAARDFLQTFRRGLLGSVMLDLDVLRGHPPAETLP------------- 355 DGP3 LARIPEVG-----AKALYNR------------------------------------------------------------YKIQLEGNCGKKFVKTLGLNLFGGDSHQAAFRILTDFRKGKFGYVSLERPPL----------------------- 375 DGP4 --LCPKDMLVTLYKIPS---------------------------------------------------------------FEAVDDFLYKVATVRGKLKKGGLVDIDAAARIVLHDWNEGKIPYYTMPPK---RDQGGHAESKIVTELAKDFNID 430 DGP5 LRRVKKEHLQRAYKIKD---------------------------------------------------------------WEDDHDFLLQLCKSSGKLLKGGEPDLMTGAKMILHDWQRGRIPFFVPPPKLDNVASESEVIVPGIDKEAIADNSQ 488 DGP6 PRHAIEDVYNISLPKPKSYEPQS-------------------------------------------------------RPPLASELLRTYCLSRGYVAS-SGLPDETRAARQILKDYIEGKLPHFAMPPE--ITRDDENETADDTLGAETREGS- 492 DGP7 PRRVIESVYNISLPKPKTYERQS-------------------------------------------------------RPPHAAELLKSYCASRGYVAS-SGLPDETKAARLILKDYIGGKLPHYAMPPG--MPQADEPDIEDTQELEDILEGS- 496

LXG__

DGP4 EVYSGESSFIGSLKTVNEFNPVIIPSNGPLNFDETMIEDESKTQTEEEAEHESDDDESMGGEEEEEAGKTKEKSETGRQNVKLYAAESMLNTKKQKAEKKKRKKAKKAGADEEDLMDGDYDFKVDYRKNKDGEDEEFQIDAKIPMAGLLPEE 582 DGP5 AAAALKAIAGIMSTQQQKDVPVQRDFYDEKDLKDDKKAKESTETDAENGTDAEEDEDAVSEDGVESDSDADEDAVSENDEEDESDSAE---------------------------------------------------------------- 576 DGP6 QTEKKGEEAPSLGLDQVLDDLSSFDLANGLVSSKTKQHKKSHRKQ----------------------------------------------------------------------------------------------------------- 537 DGP7 ESDDSAVGDETENEQVPGIDDVLDDLSSFDLANGLKSSKKVTAKKQTASHKQHKKPQRKKDRTWRVQNTEDGDGMPSVKVFQKPANTGPLTMR----------------------------------------------------------- 589

Clade1

Clade4

Clade2

Clade3

A

Figure 4

BSIN2 139-VMVVGVPNVGKSALI-153 169-TVGPLPGVTQD-180EngA1 13-VALVGRPNVGKSTLF-28 38-LVADFPGLTRD-49EngA2 213-LAIVGRPNVGKSTLT-228 238-VVYDMPGTTRD-249ThdF 219-VVIAGRPNAGKSSLL-234 242-IVTDIAGTTRD-253Era 11-FAIVGRPNVGKSTLL-26 35-ITSRKAQTTRH-46Ras 6-LVVVGAGGVGKSALT-21 27-IFVDEYDPTIE-38

DAR

G1 G2

SIN2 48-DLVIEVRDARIP-60EngA1 83-DVVLFMVDARAG-95

C

D